Project Summary: Circulating tumor cell clusters (CTCCs) represent a unique population of tumor cells, distinct from those of the primary and metastatic tumors, with significantly higher metastatic potential than circulating tumor cells (CTCs). CTCCs have recently emerged as important diagnostic and therapeutic targets, especially as a guide for the development and monitoring of personalized treatments. While microfluidic approaches have enabled significant advances in the capture of CTCCs, they still have limitations in their sensitivity, and throughput. Exogenous fluorescence based in vivo flow cytometry (IVFC) has been used to monitor CTCs and more recently CTCCs in vivo. However, this method requires an exogenous fluorescence marker and can't be translated to patients. Our goal is to establish an optimal approach for enumerating CTCCs in vivo using a combination of endogenous fluorescence (F) and light-scattering (LS) multi-wavelength information. The initial studies will focus on identification of CTCCs labeled with an exogenous fluorophore (GFP) for validation, but our aim is to detect CTCCs based on endogenous optical signals. To achieve our goal, we will perform a combination of in vitro- microfluidic based and IVFC experiments. We will acquire data at three scattering wavelengths (405, 488 and 633 nm), and two fluorescence emission bands for GFP and autofluorescence detection. We will establish data acquisition conditions that yield optimal throughput and CTCC detection sensitivity using blood samples spiked with CTCCs that are flowing at varying speeds through a range of microfluidic channel sizes (Aim 1). We will proceed with IVFC measurements using CTCC tail vein injections of GFP+ and GFP- CTCCs of two-fifteen cells to establish the sensitivity of IVFC using light scattering alone and in combination with endogenous fluorescence (Aim 2). The GFP signal and microscopic evaluations will be used as the gold standard for detection. Our sensitivity targets (higher than 65.5% for CTCCs with nine or more cells in blood) are based on the reported performance of in vitro chips optimized for CTCC detection. The proposed studies build on the extensive experience of the PI on in vivo flow cytometry and label-free imaging. In combination with our previous work, they will provide an essential foundation for establishing the potential of label-free in vivo flow cytometry for CTCC enumeration. Such a method is expected to have a significant impact in animal studies aiming to understand the role of CTCCs and the impact of new related therapies. More importantly, our expected outcomes will motivate efforts to translate the use of this technology for the study of CTCCs in humans.